1. Field of Invention
This invention relates to a method for a quantitative screening of antioxidants for potency and toxicity based on their effect on sterol superlattice formation.
2. Description of Related Art
Reactive oxygen species (ROS) are thought to play a major role in the initiation of inflammation and pathophysiological changes associated with conditions such as Alzheimer's Disease, cancer and coronary heart disease (1, 2, 3, 4, 5, 6, 7). Oxidative damage of membrane lipids by ROS should in theory be reduced or prevented by the action of lipid-soluble antioxidants. Vitamin E, beta-carotene, and ascorbyl palmitate (lipid-soluble form of vitamin C) have all been candidates in the search for a membrane-soluble supplement that would counteract the damage caused by free radicals. Recently, there has been so much conflicting information about these antioxidants that supplementation is not recommended.
Lipophilic antioxidants (e.g., α-tocopherol and ascorbyl palmitate), have received conflicting reviews regarding their value as preventative or curative agents. Initially, lipophilic antioxidants were thought to confer cardioprotective or other types of benefits; however, more recent studies have shown that these substances can be ineffective or even harmful.
Alpha-tocopherol (vitamin E) was believed to be cardioprotective (8, 9) and to be associated with a reduction of risk in prostate, breast and other types of cancers (10, 11, 12, 13), as well as with a decrease in ROS-induced damage (14). Recently, however, a number of clinical trials have dampened the initial enthusiasm about vitamin E therapy. In a meta-analysis by Miller et al., high doses of vitamin E were associated with an increase in “all-cause mortality” (15). The Women's Angiographic Vitamin and Estrogen (WAVE) Trial demonstrated no improvement in coronary angiographic findings in women on hormone replacement therapy and taking vitamins C and E when compared with placebo (16). In general, nearly all of the most recent data do not support the use of vitamin E in cardiovascular disease and cancer prevention (17).
Beta-carotene was also thought to be cardioprotective and to reduce atherosclerotic progression (18, 19). As in the case of vitamin E, beta-carotene supplementation was recommended in the prevention and treatment of cardiovascular disease, cancer and smoking-related conditions (20, 21). Again, conflicting information from more recent studies show an increased risk with beta-carotene therapy, especially in smokers (22). Several large prevention trials involving carotenoids produced disappointing and inconclusive results; the use of beta-carotene is not recommended (23).
Ascorbyl palmitate is a lipid-soluble ester of ascorbic acid (vitamin C) and palmitic acid. This compound disperses in biomembranes, but still retains the antioxidant properties of ascorbic acid (24). Although ascorbyl palmitate is a synthetic compound, it has been proposed as a mechanism to transport ascorbic acid into neural and other tissues (25, 26), and is used in many topical formulations. As with the other previously mentioned lipophilic antioxidants, different studies show ascorbyl palmitate to either protect against free radical damage (27, 28, 29, 30) or to induce further free radical damage (31, 32, 33).
It is unclear, why these lipid-soluble antioxidants may cause damage in some cases but not in others. Some hypotheses have been put forward, but there is no consensus. According to Heinecke (34), a major problem is that there is surprisingly little evidence that compounds which display antioxidant activities in vitro actually inhibit oxidative reactions in vivo. The problem is that reactive intermediates produced by the oxidation process are short-lived and difficult to monitor and quantify. For example, it would be necessary to identify a reliable marker of in vivo lipid peroxidation in order to monitor the effects of vitamin E on lipid oxidation (34). Such biochemical markers may not be readily apparent in an in vitro study. Some of the deleterious effects seen in studies of beta-carotene could have to do with the fact that the formation of beta-carotene degradation products is directly proportional to the degree of oxidative stress. These products are toxic to cells, and contribute to the increase in carcinogenic effects that are seen in smokers using beta-carotene (35). In a similar hypothesis, Meves et al. propose that the lipid component of ascorbyl palmitate probably contributes to the generation of toxic oxidized lipid metabolites (31). Finally, Pinnell discusses the possible toxicity of ascorbyl palmitate. He hypothesizes that when the ascorbyl free radical is produced in the aqueous phase (from ascorbic acid), it is relatively weak and readily reduced. However, when the free radical is produced in the membrane environment (from ascorbyl palmitate), it may cause irreversible damage before it is reduced (33). Clearly, the status of lipophilic antioxidants is in question. Until conclusive evidence exists that these compounds confer a significant benefit, their use is not recommended. The widespread disagreement over the use of lipophilic antioxidants also demonstrates the need for more fundamental studies of antioxidant-membrane interactions.
Since these antioxidants reside in the membrane, an understanding of membrane structure should provide important information about the action of a lipid-soluble antioxidant.
The presence of a lag phase by antioxidants in AAPH-induced oxidation is known in the literature (36-42). However, there are no studies that link sterol lateral organization with the potency and adverse effect of lipid-soluble antioxidants. Known screening assays have ranked antioxidants either on the basis of the antioxidant-induced lag phase, or by the ability to reduce preformed radicals. None of these previous studies used detectable sterols. They examined non-sterol probe oxidation in buffer and serum samples. Despite current developments, there is a need for a method of a quantitative screening of antioxidants for potency and toxicity.
All references cited herein are incorporated herein by reference in their entireties.